Dr. Maria Célia Jamur

THE ROLE OF MAST CELL PROTEASES IN TUMOR

ANGIOGENESIS

Dr. Maria Célia Jamur

University of São Paulo

Ribeirão Preto Medical School

Department of Cell and Molecular Biology

Local effector cells of the immune system that participate in :

Defense of the host organism

• Inflammatory reactions

• Elimination of parasites

• Innate immunity against bacterial and viral infections

MAST CELLS

EM

IgE

bacterial and viral infections

• Allergic reactions

Diseases

• Asthma

• Diseases of the central nervous system: Alzheimer’s disease

• Diseases of the cardiovascular system: Atherosclerosis

• Tumor Progression

MAST CELL FUNCTION IS RELATED TO THE RELEASE OF MAST CELL MEDIATORS

Newly Formed MediatorsPhospholipase A2 ActivationChemotactic leukotrienesProstaglandinsThromboxanesActivating Factors, PAF

FcεεεεRI

Antigen

Antigen

Preformed MediatorsGranule ReleaseHistamineHeparinEnzymesChemotactic factorsMast cell specific proteases

Newly Synthesized MediatorsTranscription Factor ActivationGrowth FactorsCytokines

TUMOR INDUCTION

1 week

INITIATION PROMOTION PROGRESSION

DMBA TPA

BALB/c mice

Diagram Modified from the German Cancer Research Center

DMBA (7,12-Dimethylbenz(a)anthracene)TPA (12-O-Tetradecanoylphorbol-13-acetate)

TUMOR MORPHOLOGY CHANGES WITH TUMOR PROGRESSION

Phase IControl

The division into morphological phases facilitates the analysis of mast cellsduring tumor progression.

Phase IIIPhase II

H&E

Keratincyst

de Souza, Jr., et al., PLoS One, 2012Sundberg, JP, et al., Mol. Carcinog., 1997

MARKERS OF TUMOR PROGRESSION INCREASE WITH TIME

Expression of ααααV and ββββ3 integrin subunits

PLC Gama Alpha Actina

de Souza, Jr., et al., PLoS One, 2012

MAST CELL NUMBERS INCREASE IN THE DERMISDURING TUMOR PROGRESSION

de Souza, Jr., et. al, PLoS One, 2012

MAST CELL SPECIFIC PROTEASES

� Exclusively expressed by mast cells and include chymases, tryptases and carboxypeptidase A

� These proteases constitute ~25% of the granule content

� Stored as active enzymes

� Implicated in several pathological conditions:

• Arthritis

• Allergic airway inflammation

• Glomerulonephritis

• Aortic aneurism

• Tumor angiogenesis Caughey, Immunol. Reviews, 2007Pejiler et al., Blood 2010da Silva et al., J.Histochem. Cytochem, 2014

PROTEASE MW CLASS TYPE ACTIVITY

Chymase ~36 kDa Serine protease mMCP-1, 2, 4 Chymotrypsin-like

MOUSE MAST CELL SPECIFIC PROTEASES

Chymase ~36 kDa Serine protease mMCP-1, 2, 4

mMCP-5

Chymotrypsin-like

Elastase

Tryptase ~36 kDa Serine protease mMCP-6, 7 Trypsin-like

Carboxypeptidase A ~50 kDa Metalloprotease mMCP-CPA Carboxypeptidase

Pejiler et al., Adv. Immunol. 2007

EXPRESSION OF MAST CELL SPECIFIC PROTEASES IS ALTERED DURING TUMOR PROGRESSION

Chymases

mMCP-4

Tryptases

mMCP-6

mMCP-5 mMCP-7

Carboxypeptidase A

CPA

de Souza, Jr. et al.,PLoS One, 2012

ENZYMATIC ACTIVITY OF CHYMASE AND TRYPTASE INCREASE WITH TUMOR

PROGRESSION

Chymase Tryptase

de Souza, Jr., et al., PLoS One, 2012

BLOOD VESSEL NUMBER AND DIAMETER INCREASE DURING TUMOR

PROGRESSION

Blood vessels immunolabeled with anti-vonWillebrand factor

Area = 1.9 mm2

Blood Vessels/Area % Field Covered Mean Vessel Diameter

de Souza, Jr., et al., PLoS One, 2012

Number of Vessels/Field* % of Field* Covered by Vessels

Correlationcoefficient (r)

Significance (p) Correlationcoefficient (r)

Significance(p)

Number of Mast Cells 0,98 0,018 0,94 0,05

THERE IS A POSITIVE CORRELATION BETWEEN BLOOD VESSELS AND MAST CELLS NUMBER AND

PROTEASE EXPRESSION

Protease Expression (r) (P) (r) (P)

mMCP-5 0,98 0,016 0,95 0,04

mMCP-6 0,92 0,07 0,97 0,02

mMCP-7 0,99 0,007 0,96 0,03

mMC-CPA 0,99 0,009 0,95 0,04

*Field = 1.9mm2

Tumor Progression

Modified from Coussens L M et al. Genes Dev. 1999;13:1382-1397

Mast Cell Proteases

Blood Vessels

IN VITRO ANGIOGENESISTUBE FORMATION ASSAY

L

Tubes

Ibidi µ-SlideAngiogenesis®

Branch pointsWimasis WimTube

rMMCP-6 AND -7 ACCELERATED TUBE FORMATION BY ENDOTHELIAL CELLS

SVEC4-10 CELLSwithout proteases

SVEC4-10 CELLSrmMCP-6

SVEC4-10 CELLSrmMCP-7

L

L

Cells were cultured for 5 hours at 37°C on Geltrex, fixed, stained with phalloidinconjugated to Alexa 488 and counterstained with DAPI .

Tube L = Loop

LL L

A few cells are spread and occasional tubes

are seen

Cells are spread and tubes and loops are

present

Tubes and loops are more prevalent

Control

rmMCP-6

rMMCP-6 AND -7 ACCELERATE TUBE FORMATION AND MATRIX PENETRATION

rmMCP-6

rmMCP-7

Cells were cultured for 5 hours at 37°C on GeltrexScanning Electron Microscopy

CO-CULTURE OF SVEC4-10 ENDOTHELIAL CELLS WITH P815 MAST CELLS INDUCES

FORMATION OF TUBES AND LOOPS

Phalloidin-Alexa 488 and DAPI

There are fewer tubes and loops than when the SEVEC4-10 cells

are cultured with proteases

Cells were cultured for 5 hours at 37°C on Geltrex

GFP-SVEC4-10 cells Tube L = LoopP815 Mast Cells

L

� Number of tubes, area covered by tubes, tube length, branching points and loops are higher when the SVEC4-10 cells are incubated with

rmMCP-7 IS MORE EFFECTIVE IN INDUCING TUBE FORMATION

Number of Tubes Tube Length

% Area Covered Branch Points

are incubated with rmMCP-7

� The average area occupied by the tubes was higher when cells were cultured with rmMCP-6

Wimasis WimTube

Mean Tube Area Loops

ANTI-mMCP-6 AND ANTI-mMCP-7 REDUCE TUBE FORMATION

Control cells were cultured in absence of antibodies or proteases

Antibodies were a gift from Dr. Michael Gurish, Brigham and Women´s Hospital, Harvard University

rmMC TRYPTASES INDUCE THE RELEASE OF ANGIOGENIC FACTORS

IL-10 CSIF

Leptin Ob

MMP-3

IGFBP-9

PDGF-AA

Platelet Factor 4

Proliferin

Serpin F1 PEDF

TIMP-4

An

gio

ge

nic

Fa

cto

rs

mMCP-7

mMCP-6

rmMCP-6

rmMCP-7

0 50 100 150 200 250

ADAMTS1

Angiopoietin-1

CXCL16

DPPIV/CD26

Endothelin-1

FGF

HB-EGF

IGFBP-2

IL-10 CSIF

Mean Optical Density

An

gio

ge

nic

mMCP-6

Controle

rmMCP-7

Control

Proteome Profiler Mouse Angiogenesis Array

CONCLUSIONS

� Mast cells are involved in the induction ofangiogenesis in the early stages of tumor development

� Mast cells modulate blood vessel growth in the� Mast cells modulate blood vessel growth in thelater stages of tumor progression

� Mast cell specific proteases play a critical role in angiogenesis by inducing release ofangiogenic factors

FMRP

Collaborators:Devandir Antonio de Souza, Jr.

Maria Rita de Cassia Campos

Vanina Danusa Toso

Ana Carolina Santana

Antonio Carlos Borges

Constance Oliver

Financial Support